1. Field Of The Invention
This invention relates to a process and materials for producing soluble biological mediators, including cytokines, lymphokines, monokines and interferons, from white blood cells. it also relates to the production of messenger RNA (mRNA) for such substances from white blood cells. More specifically, the invention relates to enhancing the ability of mitogenic and antigenic agents to induce the production of soluble biological mediators. The invention is particularly useful in producing human gamma interferon (HuIFN-.gamma.), also known as immune interferon or Type II interferon, and mRNA therefor.
2. Description of the Prior Art
As known in the art, mitogenic agents, i.e., substances that induce mitosis and cell transformation, and antigenic agents can be used to stimulate white blood cells to produce soluble biological mediators. See, for example, J. A. Georgiades, et al, "Human Immune Interferon: Purification And Activity Against a Transformed Human Cell", in Inteferon: Properties And Clinical Uses, (Khan et al, eds.), Wadley Institutes of Molecular Medicine, Dallas, Tex. 1980, pages 97-110. In particular, since as early as 1965, it has been known that the mitogen, phytohemagglutinin (PHA), an extract of red kidney beans, can be used to stimulate the production of HuIFN-.gamma. in Ieukocyte suspensions. See E. F. Wheelock "Inhibition of Virus Induced Leukemia In Mice By A Non-Tumor Virus", National Cancer Institute Monograph 22, 1965, pages 73-76; E. F. Wheelock, Science, Vol. 149, 1965, pages 310-311.
Production of soluble biological mediators in general and gamma interferon in particular from white blood cells has been notoriously difficult because of the small yields obtained by prior art methods A common element of these prior art methods has been the removal from the white blood cell population of essentially all red blood cells. This removal has typically been accomplished by one of two methods. In the more common method, ammonium chloride (NH.sub.4 Cl) has been used to lyse the red blood cells commingled with the white blood cells. See J. A. Georgiades, et al, supra. Alternatively, centrifugation in Ficoll-Hypaque gradients has been used to fractionate and thus separate the white blood cells from the red blood cells.
In direct contrast to these prior art methods, as discussed in detail below, it has been found unexpectedly that the production of soluble biological mediators and mRNA therefor can be significantly increased by the incorporation of a controlled amount of red blood cells with the white blood cells. Specifically, increases in the production of human gamma interferon on the order of 5 to 10 fold have been achieved by maintaining the ratio of red blood cells to white blood cells above about 10 to 1.